Edinburgh Research Explorer Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns Citation for published version: Llorens, C, Arcos, SC, Robertson, L, Ramos, R, Futami, R, Soriano, B, Ciordia, S, Careche, M, González- Muñoz, M, Jiménez-Ruiz, Y, Carballeda-Sangiao, N, Moneo, I, Albar, JP, Blaxter, M & Navas, A 2018, 'Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns', BMC Genomics, vol. 19, no. 1, pp. 592. https://doi.org/10.1186/s12864-018-4970-9 Digital Object Identifier (DOI): 10.1186/s12864-018-4970-9 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: BMC Genomics Publisher Rights Statement: © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 11. Oct. 2021 Llorens et al. BMC Genomics (2018) 19:592 https://doi.org/10.1186/s12864-018-4970-9 RESEARCHARTICLE Open Access Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns C. Llorens1, S. C. Arcos2, L. Robertson2, R. Ramos3, R. Futami1, B. Soriano1, S. Ciordia4, M. Careche5, M. González-Muñoz6, Y. Jiménez-Ruiz2, N. Carballeda-Sangiao6, I. Moneo6, J. P. Albar4, M. Blaxter7 and A. Navas2* Abstract Background: Anisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood. Results: Third stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes. Conclusions: A. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses. Keywords: Anisakis simplex, A. Pegreffii, Transcriptome, Allergens, Gene expression * Correspondence: [email protected] 2Departamento Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, Calle José Gutiérrez Abascal, 2, 28006 Madrid, Spain Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Llorens et al. BMC Genomics (2018) 19:592 Page 2 of 21 Background adult Anisakis from within definitive hosts is problematic, Anisakis is a genus of nematodes (Nematoda, Rhabditida, and L3 diagnosis is difficult, a molecular taxonomic ap- Ascaridomorpha: Anisakidae [1]) which parasitizes mar- proach for L3, using the nuclear ribosomal internal tran- ine mammals, fishes, molluscs and crustaceans. Anisakis scribed spacers (ITS), has been developed [20]. These nematodes complete their life cycle in the stomachs of markers have revealed both potential additional species di- cetaceans, and less frequently pinnipeds, which become versity within Anisakis as well as recombinant genotypes infected after devouring paratenic hosts (fishes, cephalo- [21–23]. The genus Anisakis is commonly accepted to in- pod molluscs or krill) harboring the arrested infective clude 10 named species: A. simplex sensu stricto (s.s.), A. third-stage larvae (L3) [2]. Humans can also be infected berlandi (formerly A. simplex C), A. pegreffii, A. typica, A. by consuming raw or undercooked fish or cephalopod ziphidarum, A. physeteris, A. brevispiculata, A. paggiae, meat. Although L3 of Anisakis spp larvae are unable to A. nascettii and A. schupakovi. At least 2 additional unde- complete their life cycle in humans, after penetration of scribed species (Anisakis sp. 1 and Anisakis sp. 2) have the human gastrointestinal tract they can cause anisakia- also been designated from L3 genotypes [24, 25]. The sis, a condition characterized by acute gastrointestinal most widely studied species are A. simplex s.s., A. pegreffii manifestations of epigastralgia, nausea, abdominal pain and A. berlandi: these constitute the Anisakis simplex and diarrhoea [3]. Allergic reactions such as sensu lato (s.l.)complex[26–28]. A. simplex s.s. and A. IgE-mediated hypersensitivity, angioedema, urticaria and pegreffii have been identified as the main etiological agents anaphylaxis are also associated with zoonotic exposure of anisakiasis [29–33] but it remains unclear if these 2 spe- of humans to Anisakis spp. [4]. Fish infected with L3 are cies are equally pathogenic. treated by freezing, which kills the larvae. However, Importantly, A. simplex s.s. and A. pegreffii are able of while infection by live L3 is necessary for many of the hybridizing in the areas where they are geographically symptoms of anisakiasis, humans sensitized to Anisakis sympatric [34, 35 ] and co-infect the same fish host [36]. may show symptoms after consuming fish that has been The described “species” with recombinant genotypes may correctly frozen, cooked and even processed [5–7]. be the products of interspecific hybridization [34]. While Many Anisakis spp. proteins maintain their allergenic no fertile adult hybrids have been found, L3 with geno- properties even after thermal treatment [7–9]. Episodes types suggestive of hybridisation between A. simplex s.s. of allergic disease in the absence of recent allergen ex- and A. pegreffii have been identified [24]. These hybrids posure have also been also described [10]. may express distinct suites of allergenic and immunoreac- Anisakiasis is emerging as an important epidemiological tive proteins [15]. problem. More than 20,000 cases of anisakiasis have been Here we present a transcriptomic analysis of A. sim- reported worldwide since 1960 [11], with higher inci- plex s.s., A. pegreffii and their hybrids sampled in the dences in areas such as Japan, the Netherlands, France, same development stage (L3) from the same host (Mer- Spain, Germany